Ribozymes represent a promising gene therapy for the treatment of AIDS. Although several different ribozymes have been shown to inhibit HIV-1 replication in vitro, important questions regarding the efficacy and safety of this approach can be best addressed in an animal model. We evaluated the ability of an asymmetric hammerhead ribozyme specific for the SIV tar sequence to inhibit viral replication in transformed cell lines. Several different promoter cassettes expressing this ribozyme were incorporated into an adeno-associated virus vector containing the selectable marker NEO (E. Bertrand, C. Carbonell, S. Chatterjee, and J. Rossi, submitted). Two constructs utilized RNA polymerase II promoter (RSV LTR and U1 snRNA) and two utilized RNA polymerase III promoters (U6 and tRNA). In addition, several of the cassettes contained motifs designed to enhance transcript stability. Stable cell lines containing these expression cassettes were generated by electroporation of vector DNA into the CEMz174 and HUT 78 cell line followed by selection in medium containing G418. Transfected cell lines were challenged at different multiplicities of infection ranging from 0.01 to 0.0001 TCID50/cell and viral replication compared with a control cell line containing a mutant ribozyme with a mutation in the active site and untransfected cell lines. We find that expression of the SIV tar ribozyme cassette under the control of the RSV and tRNA resulted in higher levels of expression than the U1 stem and U6 promoters. In Hut 78 cells, expression of the asymmetric tar ribozyme under the control of the RSV or tRNA promoters resulted in moderate inhibition of SIV replication as compared with either untransfected cells or cells transfected with the mutant ribozyme. In CEMx174 cells, expression of the asymmetric tar ribozyme under the control of the RSV or tRNA promoters also produced inhibition of SIV replication as compared with cells transfected with the mutant ribozyme. Inhibition of SIV replication in ribozyme expressing cell lines was not attributable to either decreased proliferative responses or decreased expression of CD4 (data not shown).